Role of human cytochrome P450 (CYP) in the metabolic activation of N-alkylnitrosamines: application of genetically engineered Salmonella typhimurium YG7108 expressing each form of CYP together with human NADPH-cytochrome P450 reductase

The role of human cytochrome P450 (CYP) in the metabolic activation of N-alkylnitrosamines was examined by Ames test using genetically engineered Salmonella typhimurium (S. typhimurium)YG7108 cells expressing each form of human CYP together with human NADPH-cytochrome P450 reductase (OR). The relationship between the structure of N-alkylnitrosamines and CYP form(s) involved in the activation was evaluated. Eleven strains of S. typhimurium YG7108 cells expressing each form of CYP (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 or CYP3A5) were employed. Eight N-alkylnitrosamines including N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA), N-nitrosomethylethylamine (NMEA), N-nitrosomethylpropylamine (NMPA), N-nitrosomethylbutylamine (NMBA) and N-nitrosoethylbutylamine (NEBA) were examined. Minimal concentration (MC) value of a promutagen was defined as the concentration of a chemical giving a positive result. Mutagen-producing capacity of CYP, as indicated by induced revertants/nmol promutagen/pmol CYP, for an N-alkylnitrosamine was determined for all forms of CYP. These N-alkylnitrosamines were mainly activated by CYP2E1, CYP2A6 and CYP1A1. N-alkylnitrosamines with relatively short alkyl chains such as NDMA and NMEA were primarily activated by CYP2E1 as judged by mutagen-producing capacity. With the increase of the number of the carbon atoms of the alkyl chains, the contribution of CYP2A6 increased. CYP2A6 played major roles in the activation of NDEA, NDPA, NMPA, NMBA and NEBA. Interestingly, CYP1A1 became a molecular form of CYP playing a major role in the metabolic activation of NDBA.     

Development of a Salmonella tester strain sensitive to promutagenic N-nitrosamines: expression of recombinant CYP2A6 and human NADPH-cytochrome P450 reductase in S. typhimurium YG7108

We developed a new Salmonella tester strain highly sensitive to promutagenic N-nitrosamines by introducing a plasmid carrying human cytochrome P450 2A6 (CYP2A6) and NADPH-cytochrome P450 reductase (OR) cDNA into the ada- and ogt-deficient strain YG7108. The YG7108 2A6/OR cells expressed high levels of CYP2A6 (77+/-8nmol/l) and OR (470+/-20 micromol cytochrome c reduced/min/l). The expressed CYP2A6 efficiently catalyzed coumarin 7-hydroxylation. N-Nitrosodiethylamine (NDEA), N-nitrosomethylphenylamine (NMPhA), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were mutagenic in the new strain in the absence of any exogenous activation system. The concentrations of promutagen that caused a two-fold increase in revertants were 7.1, 0.14, and 1.4 microM for NDEA, NMPhA, and NNK, respectively. YG7108 2A6/OR cells showed about 10- and 100-fold higher sensitivity to NDEA and NNK, respectively, than parental YG7108 cells assayed in the presence of rat liver S9 (final concentration, 21% (v/v)). Parental YG7108 cells did not detect NMPhA mutagenicity even in the presence of rat liver S9. We believe that this is the first demonstration that CYP2A6 is responsible for the metabolic activation of NMPhA. The established tester strain may be useful to predict human activation of N-nitrosamine promutagens.     

Establishment of ten strains of genetically engineered Salmonella typhimurium TA1538 each co-expressing a form of human cytochrome P450 with NADPH-cytochrome P450 reductase sensitive to various promutagens

We newly developed 10 Salmonela typhimurium TA1538 strains each co-expressing a form of human cytochrome P450s (P450 or CYP) together with NADPH-cytochrome P450 reductase (CPR) for highly sensitive detection of mutagenic activation of mycotoxins, polycyclic aromatic hydrocarbons, heterocyclic amines, and aromatic amines at low substrate concentrations. Each form of P450 (CYP1A1, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 or CYP3A5) expressed in the TA1538 cells efficiently catalyzed the oxidation of a representative substrate. Aflatoxin B1 was mutagenically activated effectively by CYP1A1, CYP1A2, and CYP3A4 and weakly by CYP2A6 and CYP2C8 expressed in S. typhimurium TA1538. CYP1A1 and CYP1A2 were responsible for the mutagenic activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-acetylaminofluorene. Benzo[a]pyrene was also activated efficiently by CYP1A1 and weakly by CYP1A2, CYP2C9, CYP2C19, and CYP3A4 expressed in TA1538. These results suggest that the newly developed S. typhimurium TA1538 strains are applicable for detecting the activation of promutagens of which mutagenic activation is not or weakly detectable with N-nitrosamine-sensitive YG7108 strains expressing human P450s.     

Inhibition by green tea catechins of metabolic activation of procarcinogens by human cytochrome P450

Catechins, major polyphenol constituents of green tea, are potent chemopreventive agents against cancers caused by chemical carcinogens in rodents. The effects of four epicatechin derivatives, epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC) and epicatechin (EC), on the metabolic activation of benzo[a]pyrene (B[a]P), 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) and aflatoxin B(1) (AFB(1)) by human cytochrome P450 (CYP) were examined. B[a]P, PhIP and AFB(1) were activated by respective human CYP1A1, CYP1A2 and CYP3A4 expressed in the membrane fraction of genetically engineered Salmonella typhimurium (S. typhimurium) TA1538 cells harboring the human CYP and human NADPH-CYP reductase (OR), when the membrane fraction was added to S. typhimurium TA98. Galloylated catechins, ECG and EGCG inhibited the mutagenic activation potently, while EGC and EC showed relatively weak inhibitory effects. Catechins also inhibited the oxidations of typical substrates catalyzed by human CYPs, namely ethoxycoumarin O-deethylation by CYP1A1, ethoxyresorufin O-deethylation by CYP1A2 and midazolam 1'-hydroxylation by CYP3A4. The IC(50) values of catechins for the inhibition of human CYP were roughly the same as those seen in the mutagenic activation. EGCG inhibited other forms of human CYP such as CYP2A6, CYP2C19 and CYP2E1, indicating the non-specific inhibitory effects of EGCG toward human CYPs. Furthermore, EGCG inhibited human NADPH-cytochrome CYP reductase (OR) with a K(i) value of 2.5 microM. These results suggest that the inhibition of the enzyme activity of CYP is accounted for partially by the inhibition of OR.     

Development of a new genotoxicity test system with Salmonella typhimurium OY1001/1A2 expressing human CYP1A2 and NADPH-P450 reductase.

In order to develop a new tester strain detecting environmental promutagens and procarcinogens, we introduced two plasmids into Salmonella typhimurium TA1535; one contains the cDNAs of human cytochrome P450 (P450 or CYP) 1A2 and NADPH-P450 reductase and the other (pOA101) a umuC"lacZ fusion gene. The newly developed tester strain, S. typhimurium OY1001/1A2, was found to express P450 at a level of 0.15 nmol/ml in whole cell culture. Membrane fractions, when isolated from this tester strain, contained 0.04 P450 nmol/mg protein and a reductase activity of 170 nmol cytochrome c reduced/min/mg protein and were active in catalyzing CYP1A2-dependent 7-ethoxyresorufin O-deethylation and metabolic activation of heterocyclic aromatic amines to DNA-damaging products in a conventional tester S. typhimurium NM2009 strain, only when NADPH was added as a reducing equivalent. In the OA1002/1A2 strain, heterocyclic aromatic amines (e.g., IQ, MeIQ, and MeIQx) were found to be activated to reactive metabolites that cause induction of umuC gene expression in a dose-dependent manner, without addition of external NADPH. These results indicate that the newly established strain can be of use to detect mutagenic and carcinogenic potencies of environmental chemicals without addition of metabolic activation system.     

Mutagenic activation of betel quid-specific N-nitrosamines catalyzed by human cytochrome P450 coexpressed with NADPH-cytochrome P450 reductase in Salmonella typhimurium YG7108

Betel quid chewing is known to cause cheek cancer in a wide area covering Africa to Asia. Areca nut contained in the betel quid is believed to give rise to carcinogenic N-nitrosamines. In the present study, the roles of human cytochromes P450 (P450 or CYP) in the mutagenic activation of betel quid-specific N-nitrosamines such as 3-(N-nitrosomethylamino)propionitrile (NMPN), 3-(N-nitrosomethylamino)propionaldehyde (NMPA) and N-nitrosoguvacoline (NG) were examined by using genetically engineered Salmonella typhimurium YG7108 expressing each form of human P450 together with NADPH-P450 reductase, which had been established in our laboratory. Among typical P450s (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2A13, CYP2D6 or CYP3A4) examined, CYP2A6 was the most efficient activator of NMPN, followed by CYP1A1 and CYP1B1. The mutagenic activation of NMPN by CYP2A6 was seen at the substrate concentrations of microM levels (approximately 100 microM). The activation of NMPA was catalyzed predominantly by CYP2A13 and to lesser extents by CYP2A6, CYP1A1, CYP1A2 and CYP1B1. The activation of NMPA by CYP2A13 was detectable at the substrate concentrations of microM levels (approximately 1 microM). NG was activated by CYP2A13 and CYP2A6, the genotoxicity of NG being much lower than that of NMPA or NMPN. Based on these data, we conclude that human CYP2A subfamily members play important roles in the mutagenic activation of essentially all betel quid-related N-nitrosamines tested in the present study.     

CYP2A6 gene deletion reduces susceptibility to lung cancer.

CYP2A6 is an enzyme with a high ability to activate a nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), to its potent and ultimate carcinogen. In the present study, we investigated the relationship between genetic polymorphism of CYP2A6 and lung cancer risk in a case-control study of Japanese subjects. Genotyping of the CYP2A6 gene in both healthy volunteers and lung cancer patients was conducted. The frequency with which the subjects carried homozygotes of the CYP2A6 gene deletion-type mutation (deletion), which causes lack of the enzyme activity, was lower in the lung cancer patients than in the healthy control subjects. The odds ratio (OR) of the group homozygous for the deletion was significantly lower and calculated to be 0.25 (95% CI; 0.08-0.83) when the OR for the population with homozygotes of the CYP2A6 wild-type gene was defined as 1.00. In the allelic-base analysis, there was also a significant decrease in the OR for the deletion allele. These data suggest that deficient CYP2A6 activity due to genetic polymorphism reduces lung cancer risk.     

Genotoxic activation of 2-phenylbenzotriazole-type compounds by human cytochrome P4501A1 and N-acetyltransferase expressed in Salmonella typhimurium umu strains

Four 2-phenylbenzotriazole (PBTA)-type compounds (PBTA-4, PBTA-6, PBTA-7, and PBTA-8) were identified as major mutagens in blue cotton/rayon-adsorbed substances collected at sites below textile dyeing factories or municipal water treatment plants treating domestic waste and effluents from textile dyeing factories in several rivers in Japan. The main purpose of this study is to understand the basis of the roles of human cytochrome P450 (CYP) and N-acetyltransferases (NATs) in genotoxic activation of PBTA derivatives. We compared the induction of umuC gene expression as a measure of genotoxicity using Salmonella typhimurium TA1535/pSK1002 (parental strain), NM2009 (bacterial O-acetyltransferase-overexpressing strain) established in our laboratories. PBTA-4, PBTA-6, PBTA-7, and PBTA-8 induced the umuC gene expression more strongly in the bacterial O-acetyltransferase-overproducing strain than in the parental strain in the presence of rat S9 mix. We determined the activation of PBTA derivatives by cDNA-based recombinant (Trichoplusia ni) systems expressing human or rat cytochrome P450 enzymes (P450 or CYP) and NADPH-P450 reductase using S. typhimurium NM2009. The results showed that human recombinant CYP1A1 enzyme was much more active than CYP1A2 and CYP3A4 in the genotoxic activation of PBTA-4, PBTA-6, PBTA-7, and PBTA-8. Similarly, rat recombinant CYP1A1 enzyme catalyzed the activation of these chemicals at high rates. alpha-Naphthoflavone, a known inhibitor of CYP1A1, was found to inhibit genotoxic activation caused by PBTA derivatives. We further determined the activation of PBTA derivatives using S. typhimurium NM6001 (human NAT1-expressing strain), S. typhimurium NM6002 (human NAT2-expressing strain), and S. typhimurium NM6000 (O-AT-deficient parent strain) in the presence of S9 mix. PBTA-4 showed almost similar sensitivity in the NAT1-expressing strain and the NAT2-expressing strain, although NAT2-expressing strain exhibited relatively higher sensitivity to PBTA-6, PBTA-7, and PBTA-8 than NAT1-expressing strain. The results support the view that O-acetylation by human NAT1 and NAT2 enzymes is involved in the genotoxic activation of PBTA compounds. These results demonstrate for the first time that human P4501A1 and NATs (NAT1 and NAT2) contribute significantly to the activation of PBTA-type compounds to genotoxic metabolites that induce umuC gene expression in S. typhimurium tester strains.     

Genetic polymorphism of cytochrome P450 2E1 in the Turkish population.

CYP 2E1 is involved in metabolic activation of carcinogenic N-nitrosamines, benzene, urethane and other low molecular weight compounds. CYP2E1 gene is present in the population in various polymorphic forms. We detected the RFLP of the human CYP2E1 gene with the restriction endonuclease PstI, RsaI and DraI in a group of 153 Turkish individuals. According to the results of the PstI/RsaI analysis, 96.07% of the subjects were of the c1/c1 genotype, and 3.93% were of the c1/c2 genotype. In the DraI RFLP analysis, 84.30% DD genotype, 15.03% CD genotype and 0.66% CC genotype were determined. The data obtained may be useful in epidemiological studies of the influence of CYP2E1 polymorphism on carcinogenesis.     

Modulation of human dUTPase using small interfering RNA

Analyzing the CYP2A6 gene of subjects who showed a poor metabolic phenotype toward SM-12502, we discovered a novel mutant allele (CYP2A6*4C) lacking the whole CYP2A6 gene. Using genetically engineered Salmonella typhimurium expressing a human CYP, we found that CYP2A6 was involved in the metabolic activation of a variety of nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) contained in tobacco smoke. Taking these results into consideration, we hypothesized that the subjects carrying the CYP2A6*4C allele had lower risk of tobacco-related lung cancer. In accordance with our hypothesis, our epidemiological studies indicated that smokers homozygous for the CYP2A6*4C allele showed much lower odds ratios toward cancer risk. Other mutant alleles reducing the CYP2A6 activity, besides CYP2A6*4C, also reduced the risk of lung cancer in smokers, particularly of squamous-cell carcinoma and small-cell carcinoma, both smoking-related cancers. 8-Methoxypsoralen, an inhibitor of CYP2A6, efficiently prevented the occurrence of adenoma caused by NNK in A/J mice.     

Metabolic activation of heterocyclic amines and other procarcinogens in Salmonella typhimurium umu tester strains expressing human cytochrome P4501A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4 and human NADPH-P450 reductase and bacterial O-acetyltransferase

We investigated roles of different forms of cytochrome P450 (P450 or CYP) in the metabolic activation of heterocyclic amines (HCAs) and other procarcinogens to genotoxic metabolite(s) in the newly developed umu tester strains Salmonella typhimurium (S. typhimurium) OY1002/1A1, OY1002/1A2, OY1002/1B1, OY1002/2C9, OY1002/2D6, OY1002/2E1 and OY1002/3A4, which express respective human P450 enzymes and NADPH-cytochrome P450 reductase (reductase) and bacterial O-acetyltransferase (O-AT). These strains were established by introducing two plasmids into S. typhimurium TA1535, one carrying both P450 and the reductase cDNA in a bicistronic construct under control of an IPTG-inducible double tac promoter and the other, pOA102, carrying O-AT and umuC"lacZ fusion genes. Expression levels of CYP were found to range between 35 to 550 nmol/l cell culture in the strains tested. O-AT activities in different strains ranged from 52 to 125 nmol isoniazid acetylated/min/mg protein. All HCAs tested, and 2-aminoanthracene and 2-aminofluorene exhibited high genotoxicity in the OY1002/1A2 strain, and genotoxicity of 2-amino-3-methylimidazo [4,5-f]quinoline was detected in both the OY1002/1A1 and OY1002/1A2 strains. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]-indole and 3-amino-1-methyl-5H-pyrido[4,3-b]-indole were activated in the OY1002/1A1, OY1002/1B1, OY1002/1A2, and OY1002/3A4 strains. Aflatoxin B(1) exhibited genotoxicity in the OY1002/1A2, OY1002/1A1, and OY1002/3A4 strains. beta-Naphthylamine and benzo[a]pyrene did not exhibit genotoxicity in any of the strains. These results suggest that CYP1A2 is the major cytochrome P450 enzyme involved in bioactivation of HCAs.     

Polymorphisms in detoxification genes CYP1A1, CYP2E1, GSTT1 and GSTM1 in gastric cancer susceptibility

Cytochrome P450 (CYP) and glutathione S-transferase (GST) enzymes are involved in activation and detoxification of many potential carcinogens. Genetic polymorphisms in those enzymes have been found to influence the interindividual susceptibility to cancer. Some polymorphisms of those enzymes have been associated specifically with susceptibility to gastric cancer. We conducted a study in a Costa Rican population, where gastric cancer incidence and mortality rates are among the highest in the world. We investigated whether such variations affected the risk of developing gastric cancer. Subjects included 31 with gastric cancer, 58 controls with gastric injures others than cancer and 51 normal controls confirmed by X-rays (double-contrast) or endoscopic diagnostic. DNA from peripheral white blood cell was obtained from all subjects. Deletion of GSTT1 and GSTM1 was assessed by multiplex PCR and genotyping of CYP2E1 was performed using a PCR-based restriction fragment length polymorphism assay with the restriction enzyme PstI and the gene CYP1A1 using the restriction enzyme MspI The prevalence of CYP1A1 Msp1 polymorphism, GSTT1 and GSTM1 null genotype was similar in the three groups of individuals (p = 0.73, p = 0.88 y p = 0.89 respectively). Our findings suggest that the polymorphism CYP2E1 PstI could be associated with a reduced risk of having gastric cancer (OR = 0.09, IC95%:0.01 - 0.83).     

Association analysis of gene polymorphism in alcohol metabolizing enzymes with risk for coronary atherosclerosis

The allele and genotype distribution of two alcohol dehydrogenase genes ADH1B (exon 3 polymorphism A/G (47His)), ADH7 (intron 5 polymorphism G/C) and cytochrome P450 2E1 gene (CYP2E1; 5'-flanking region G/C and intron 6 T/A polymorphisms) were examined in Russian (Tomsk, n = 125) healthy population and in coronary atherosclerosis patients (CA, n = 92). The genotype frequencies followed the Hardy-Weinberg equilibrium and the alleles were in linkage equilibrium or gametic equilibrium in the control sample. Only two CYP2E1 gene polymorphisms were in linkage disequilibrium. The frequencies of the derived alleles at ADH1B (*G (+MslI) allele), CYP2E1 (**C2 (+PstI) allele) and CYP2E1 (*C (-Dra I)2 allele) were 8.48 +/- 1.86%; 1.20 +/- 0.69% and 10.00 +/- 1.90%, respectively. The 2ADH7 gene polymorphism showed a high level of heterozygosity; the frequency of the ADH7*C (-Sty I) allele was 44.58 +/- 3.21%. A significantly higher frequency of CYP2E1 (*C2 (+Pst I)) allele has been revealed in the CA group (P = 0.043; OR = 4.23; 95% CI 1.03-20.01). The tendency to significant effect of A1A2 genotype in ADH1B Msl 1 polymorphism was observed for systolic blood pressure in the control group (P = 0.068). The statistically significant two-way interaction effects of ADH7 StyI and CYP2E1 DraI on diastolic blood pressure (P = 0.029) and on the serum high density lipoprotein level (P = 0,042) were also revealed. Association of A1A2 genotype in ADHIB Msl I polymorphism with reduced amount in a serum of a very low density lipoprotein level (P = 0.045) have also been shown. This may result from multifunctional activity of alcohol metabolizing enzymes and their involvement in many metabolic and free radical reactions in the body.     

Mutagenicity of nitrosamines in methyltransferase-deficient strains of Salmonella typhimurium coexpressing human cytochrome P450 2E1 and reductase

Although dialkylnitrosamines are environmentally significant carcinogens, the use of short-term bioassays to assess the mutagenic potential of these compounds is problematic. The Ames test, a mutagenicity assay based on the reversion of Salmonella typhimurium histidine auxotrophs, is the most widely used bioassay in genetic toxicology, but the traditional Ames tester strains are largely insensitive to dialkylnitrosamine mutagenicity. We have constructed two mutagenicity tester strains that co-express full-length human cytochrome P450 2E1 and P450 reductase in S. typhimurium lacking ogt and ada methyltransferases (YG7104ER, ogt- and YG7108ER, ogt-, ada-). These new strains are susceptible to dialkylnitrosamine mutagenicity in the absence of an exogenous metabolic activating system (S9 fraction). Mutagenicity is dependent upon the coexpression of P450 2E1 with P450 reductase and is similar to or greater than that obtained with the parental strains in the presence of S9 fraction from ethanol-induced rat liver. These strains were also sensitive to nitrosamines with longer alkyl side chains including diethylnitrosamine, dipropylnitrosamine and dibutylnitrosamine. Mutagenicity decreased with alkyl chain length, consistent with the stringency of the ada-encoded enzyme for methyl and ethyl DNA adducts. These new strains may prove useful in the evaluation of nitrosamine contamination of food and environmental samples.     

The gene CYP3 encoding P450pcn1 (nifedipine oxidase) is tightly linked to the gene COL1A2 encoding collagen type 1 alpha on 7q21-q22.1.

CYP3, the gene which encodes the hepatic cytochrome P450pcn1, the isozyme responsible for the metabolic oxidation of the calcium channel-blocking drug nifedipine, has recently been mapped to human chromosome 7 using somatic cell hybrids. Using multilocus linkage analysis in CEPH families, we examined the linkage of a cDNA probe (hPCN1) for CYP3 to the oncogene MET, the pro-alpha 2(1) collagen gene COL1A2, and the T-cell receptor beta-chain gene TCRB, together with three arbitrary loci D7S8, D7S13, and D7S16, defined by the anonymous DNA probes pJ3.11, pB79a, and p7C22, respectively. From 70 CEPH parents screened with a StyI RFLP for hPCN1, four informative families were found each with both parental and maternal grandparents and 6-11 children per family. Tight linkage emerged between CYP3 and COL1A2, with a maximum combined lod score of 5.72 at theta = 0, suggesting the most likely subchromosomal localization of CYP3 is 7q21.3-q22.1.     

Deletion of the entire cytochrome P450 CYP2D6 gene as a cause of impaired drug metabolism in poor metabolizers of the debrisoquine/sparteine polymorphism.

The debrisoquine/sparteine polymorphism is associated with a clinically important genetic deficiency of oxidative drug metabolism. From 5% to 10% of Caucasians designated as poor metabolizers (PMs) of the debrisoquine/sparteine polymorphism have a severely impaired capacity to metabolize more than 25 therapeutically used drugs. The impaired drug metabolism in PMs is due to the absence of cytochrome P450IID6 protein. The gene controlling the P450IID6 protein, CYP2D6, is located on the long arm of chromosome 22. A pseudogene CYP2D8P and a related gene CYP2D7 are located upstream from CYP2D6. This gene locus is highly polymorphic. After digestion of genomic DNA with XbaI endonuclease, restriction fragments of 11.5 kb and 44 kb represent mutant alleles of the cytochrome CYP2D6 gene locus associated with the PM phenotype. In order to elucidate the molecular mechanism of the mutant allele reflected by the XbaI 11.5-kb fragment, a genomic library was constructed from leukocyte DNA of one individual homozygous for this fragment and screened with the human IID6 cDNA. The CYP2D genes were isolated and characterized by restriction mapping and partial sequencing. We demonstrate that the mutant 11.5-kb allele results from a deletion involving the entire functional CYP2D6 gene. This result provides an explanation for the total absence of P450IID6 protein in the liver of these PMs.     

Cytochrome P450 2A6 deletion polymorphism and risk of lung cancer: a meta-analysis

Previous studies concerning the association between cytochrome P450 2A6 (CYP2A6) deletion polymorphism and lung cancer risk provided controversial results. To clarify the precise association, a meta-analysis was performed. The electronic databases PubMed, Chinese Biomedical Database and Chinese National Knowledge Infrastructure Database were searched for case–control studies last updated on June 3, 2012 that investigated CYP2A6 deletion polymorphism and lung cancer risk. The odds ratio (OR) and its respective 95 % confidence interval (95 % CI) were used to measure the strength of association by means of a genetic model free approach. A total of 8 studies including 2,607 cases and 2,595 controls met the inclusion criteria and were subjected to the final analysis. The most appropriate co-dominant model was adopted. Overall, we found that CYP2A6 *1/*1 genotype was associated with an increased risk of lung cancer relative to *4/*4 genotype (OR = 2.65, 95 % CI: 1.84–3.81, P < 0.001). Significant association was also detected among Asians. Publication bias was absent in this meta-analysis. Therefore, our data suggested that the presence of the CYP2A6 *1/*1 might be associated with an increased lung cancer risk, especially for Asians. Further studies well-designed among different ethnicity populations are required.     

Association of CYP2A6*4 with Susceptibility of Lung Cancer: A Meta-Analysis

ObjectivesTo assess the association between the variant of Cytochrome P450 2A6 whole gene deletion (CYP2A6*4) polymorphism and risk of lung cancer.MethodsTwo investigators independently searched the PubMed, Elsevier, EMBASE, Web of Science, Wiley Online Library and Chinese National Knowledge Infrastructure (CNKI). Pooled odds ratios (ORs) and 95% confidence intervals (95% CIs) for CYP2A6*4 and lung cancer were calculated in a fixed-effects model (the Mantel-Haenszel method) and a random-effects model (the DerSimonian and Laird method) when appropriate.ResultsThis meta-analysis included seven eligible studies, which included 2524 lung cancer cases and 2258 controls (cancer–free). Overall, CYP2A6*4 was associated with the risk of lung cancer (allele*4 vs. allele non-*4, pooled OR  = 0.826, 95% CI  = 0.725−0.941, P-value  = 0.004). When stratifying for population, significant association was observed in Asian (additive model, pooled OR  = 0.794, 95% CI  = 0.694−0.909, P-value  = 0.001; dominant model, pooled OR  = 0.827, 95% CI  = 0.709−0.965, P-value  = 0.016; recessive model (pooled OR  = 0.444, 95% CI  = 0.293−0.675, P-value <0.0001). In the overall analysis, a comparably significant decrease in the frequency of *4/*4 genotype was detected between cases and controls in Asian while no *4/*4 genotype was detected in Caucasian in collected data.ConclusionThis meta-analysis suggests that the CYP2A6*4 polymorphism is associated with susceptibility of lung cancer in Asian. The whole gene deletion of CYP2A6 may decrease the risk of lung cancer in Asian samples.     

Effects of exposure to diesel exhaust particles (DEP) on pulmonary metabolic activation of mutagenic agents

Exposure of rats to diesel exhaust particles (DEP) or carbon black (CB) has been shown to induce time-dependent changes in CYP1A1and CYP2B1 in the lung. The present study evaluated the role of these metabolic enzymes on the pulmonary bioactivation of mutagens. Male Sprague-Dawley rats were intratracheally instilled with saline (control), DEP or CB (35 mg/kg body weight) and sacrificed at 1, 3, or 7 days post-exposure. Both control and exposed lung S9 increased the mutagenic activity of 2-aminoanthracene (2-AA), 2-aminofluorene (2-AF), 1-nitropyrene (1-NP), and the organic extract of DEP (DEPE) in Ames tests with Salmonella typhimurium YG1024 in a dose-dependent manner. Lung microsomes prepared form control or particle-exposed S9, but not cytosolic protein, activated 2-AA mutagenicity. Compared to saline controls, CB-exposed S9 was a less potent inducer of 2-AA mutagenicity at all time points, whereas DEP-exposed S9 was less potent than control saline at 3 and 7 days but not 1 day post-exposure. At 3 days post-exposure, DEP- or CB-exposed lung S9 did not significantly affect the mutagenicity of DEPE or 1-NP, when compared to the controls. The mutgenicity of 2-AA, 2-AF, 1-NP, and DEPE were significantly decreased in the presence of inhibitors for CYP1A1 (alpha-naphthoflavone) or CYP2B (metyrapone), but markedly enhanced by CYP1A1 or CYP2B1 supersomes with all the cofactors, suggesting that both CYP1A1 and CYP2B1 were responsible for mutagen activation. These results demonstrated that exposure of rats to DEP or CB altered metabolic activity of lung S9 and S9 metabolic activity dependent mutagen activation. The bioactivation of mutagens are metabolic enzyme- and substrate-specific, and both CYP1A1 and CYP2B1 play important roles in pulmonary mutagen activation.     

Frequencies of four genetic polymorphisms in the CYP1A2 gene in Turkish population

Cytochrome P450 (CYP) 1A2 gene is involved in the metabolic activation of several carcinogens and altered metabolization of some clinically used drugs. We aimed to investigate the distributions of genetic polymorphisms -3860 (G/A)(CYP1A2*1C) and -2467 (T/del)(CYP1A2*1D) in the 5'-flanking region and -739 (T/G)(CYP1A2*1E) and -163(C/A)(CYP1A2*1F) in the first intron of the CYP1A2 gene in 110 unrelated healthy Turkish volunteers by PCR-RFLP technique. The frequencies of each polymorphism in Turkish population were found as 0.04, 0.92, 0.01, 0.27 for CYP1A2*1C, CYP1A2*1D, CYP1A2*1E, CYP1A2*1F, respectively. Compared with other populations, CYP1A2*1D has been found to be significantly increased in Turkish population. On the other hand, in general, the frequencies of the other polymorphisms were concordant with those in the Egyptian and Caucasian populations, and were different from those in the Japanese, Chinese and Ethiopian populations. Our results suggest that due to increased frequency of CYP1A2*1D in Turkish population, functional significance of CYP1A2*1D should be evaluated. It might be screened to determine the relationship between CYP1A2*1D and CYP1A2 related drug metabolisms in associated groups.